Generally this invention relates to droplet flow cytometers such as are used for the analysis and sorting of substances contained within separate droplets. Specifically, the invention relates to aspects of such systems which act to form regular droplets after exit from a nozzle orifice.
Droplet flow cytometers have been in clinical and research use for many years. Basically, the systems act to position small amounts of a substance within individual droplets of a sheath fluid. These droplets can be made uniform by utilizing an oscillator which emits a predominant frequency. These oscillations are usually applied to the nozzle container. Since droplet flow cytometry is heavily utilized in both research and clinical environments, such systems have been the subject of much refinement. One of the facets of these systems which has been particularly challenging, however, is the aspect of controlling the drop formation. As to this aspect it has not only been difficult to practically achieve processing rates of much more than 40 kilohertz, it has also been difficult to deal with the incidents of using relatively high power to drive the oscillators involved.
It should be noted that each of the challenges faced in the field of droplet formation for flow cytometers is largely unique to that field. Even seemingly similar fields such as those involving channel-type flow cytometers are not very analogous as they do not face such problems. Their operation as continuous flow devices rather than droplet formation devices makes much of the understandings available in that field inapplicable to the challenges and problems faced in flow cytometry droplet formation systems.
To some degree the challenges for droplet formation may be the result of the fact that although drop formation has been modeled with significant theoretical detail, in practice it still remains a somewhat empirical subject. While on one level exhaustive mathematical predictions are possible, in practice these predictions can be greatly tempered—and are often revised—by the fact that materials limitations, inherent substance variations, and the like contribute heavily to the end result. A number of “advances” in this field have even proved to be either unnecessary or unworkable in practice.
The level of oscillation energy required in order to achieve uniform droplet formation has, prior to the present invention, been very subject to empirical constraints. This power (often expressed as a voltage amplitude applied to a piezoelectric crystal oscillator) has previously been in the ten volt range. Unfortunately, this relatively high voltage not only results in a need for more robust circuitry, but it also has the undesirable practical consequence of resulting in undesirable electromagnetic emissions. These emissions can impact the sensitivity of the flow cytometer or other nearby equipment. Further, as the desire for higher processing frequencies is pursued, this problem is compounded. Although these problems have been know for years, prior to the present invention it has apparently been an accepted attitude that in order to achieve higher frequencies, still higher oscillation energies are a physical requirement. This invention proves this expectation to be untrue. An example of the extremes to which this rational had been applied is shown in U.S. Pat. No. 4,361,400 to Gray where droplet formation frequencies in the range of 300 to 800 kilohertz had been achieved. This design had required an oscillator powered by approximately 80 volts. The apparent physical requirement of higher powers in order to achieve higher droplet frequencies may have been one reason that most practical droplet flow cytometers operated only in the range of 10 to 50 kHz. The present invention shows that such a relationship is not a physical requirement and, in fact, shows that droplet formation speeds in the 100-200 kHz range are actually possible with only millivolts of power applied to an oscillator.
Yet another problem practically encountered in this field was the challenge of resonances existing within the nozzle assembly. Again, this appears to have simply been accepted as a necessary incident of workable systems and may have resulted in an attitude among those having ordinary skill in the art that it was not practical to vary frequency without unacceptable changes in the performance of the entire system. There also seems to have been some confusion as to the appropriate way to apply the droplet forming oscillations. U.S. Pat. No. 4,302,166 shows that the oscillations are applied to the nozzle container perpendicular to the fluid flow, whereas, U.S. Pat. No. 4,361,400 suggests applying the oscillations to the nozzle container parallel to the lines of flow. In fact, the present invention discloses that each of these systems are suboptimal in that they may even act to generate the resonances and variations in frequency response of the nozzle system.
An even more paradoxical situation exists with respect to the problem of maintaining laminar flow within the nozzle system of a droplet flow cytometer. Although those having ordinary skill in this field have known for years that maintaining laminar flow was desirable, until the present invention, practical systems utilizing replacement tips have not been optimally designed so as to achieve the goal of truly laminar flow. For instance, U.S. Pat. No. 4,361,400 as well as the 1992 publication by Springer Laboratory entitled “Flow Cytometry And Cell Sorting”, each show replaceable nozzle tip designs in which laminar flow is disrupted at the junction between the nozzle body and the nozzle tip. Again, such designs seem to present almost a paradox in that they obviously are not optimum from perspective of a goal which has long been known as those having ordinary skill in the art. The present invention not only recognizes this goal but also demonstrates that a solution has been readily available.
Yet another problem encountered in this field is the need to vary parameters to optimize actual conditions encountered in processing. Again theory and practice did not mix well. While systems were usually designed for optimum conditions, in actual usage such conditions rarely existed. Thus, as U.S. Pat. No. 4,070,617 recognized, designs which allow variation of the substance output velocity within the sheath fluid were desirable. Although such systems permitted some variation, it was recognized that such variations necessarily made conditions within the flow cytometer suboptimal for the simple reason that there is a very definite physical relationship between the sheath substance and drop parameters which must be maintained. Since these parameters are well known to those having ordinary skill in the art (as also indicated in U.S. Pat. No. 4,302,166), the variations required in practice appear to have been accepted as a necessary evil. To some extent, the resulting reduced resolution appears to have been accepted without question. Again, the present invention realizes that approaches which moved conditions away from optimal were not a necessary incident of adapting to conditions practically encountered; it shows that solutions which allow for variation and yet maintain optimal flow conditions are possible.
As explained, most of the foregoing problems had long been recognized by those having ordinary skill in the art. Solutions, however, had either been perceived as unlikely or not been recognized even though the implementing elements had long been available. This may also have been due to the fact that those having ordinary skill in the art may not have fully appreciated the nature of the problem or may have been due to an actual misunderstanding of the physical mechanisms involved. These appear to have included the misunderstanding that actually moving the nozzle was the proper way to induce the droplet forming oscillations and the simple failure to realize that it was possible to coordinate the desire for replaceable nozzle tips with the desire for laminar flow within the flow cytometer nozzle assembly. Similarly, those skilled in the art had long attempted to achieve higher frequency systems which were practically implementable and had attempted to achieve variations which would to the largest extent possible maintain optimal conditions. Their attempts often led them away from the technical directions taken by the present invention and may even have resulted in the achievements of the present invention being considered an unexpected result of the approach taken.